Congenital heart defects (CHDs) afflict 36,000 babies born in the US each year and survivors often require several surgical interventions in their shortened lifetime. Despite continuous efforts, the mechanisms leading to CHDs remain largely unclear. In part, this is because most developmental cardiology studies fail to address the influential role of altered cardiac function in cardiogenesis. Myocardial excitation, cardiomyocyte contraction and hemodynamics are all likely to modify expression pathways responsible for heart development. However, due to the lack of proper imaging tools, these biomechanical and electrophysiological signals are not fully identified or well understood especially at early loopin stages, when the trajectory to heart defects can begin. In the previous funding period, we developed and demonstrated 4-D optical coherence tomography (OCT) technology as a powerful tool to image contraction dynamics and hemodynamics in the early looping heart. In this proposed project period, we will complement OCT with optical mapping (OM) using potentiometric and calcium-sensitive fluorescent dyes to reveal patterns of cardiac conduction and calcium signaling. With an integrated imaging system that can capture contraction mechanics, hemodynamics, calcium transients, and electrical conduction throughout the developmental stages during which the heart tube is looping, we will be able to investigate these individual factors, the complex interplay between them and their role in the emergence of CHDs. The integration of OCT and OM will support and enable new technology and experimental methods. We will utilize OCT to correct motion during OM imaging, obviating the need for excitation-contraction (E-C) decoupling drugs, thus enabling E-C coupling to be studied directly and comprehensively for the first time in the developing heart. We will develop 3-D OM using second-harmonic generating potentiometric dyes will and optical coherence microscopy, enabling discrimination of the 3-D heterogeneity of the conduction system of the radially asymmetric embryonic heart tube. We will develop in vivo calcium imaging by infecting quail embryos causing them to genetically express a Ca++-sensitive protein. These new tools may be applied to numerous experimental models such as ablation (e.g. neural crest cells, NCCs), environmental perturbation (e.g. hypoxia), or chemical exposure (e.g. alcohol). We have chosen to focus on the connections between the etiology of CHDs associated with Fetal Alcohol Syndrome (FAS), NCCs, and their ability to influence cardiac function from an early stage. Based on the literature and our own preliminary findings, we propose that ethanol exposure in the embryonic model at a vulnerable stage disrupts NCC development, and leads to heart defects through the impairment of cardiac function. We also hypothesize that a combination of folate/myo-inositol (FA/MI) will alleviate these functional abnormalities, and prevent FAS- related CHDs. Our proposed studies could comprise a first step toward developing new therapeutic strategies based on FA/MI prevention of birth defects to benefit public health.